Nonintegrating Lentiviral Vectors Towards Clinical Trials

描述(申请人提供)：非整合慢病毒载体(NILV)提供了一种降低未分裂细胞中插入突变风险的方法，并使潜在危险基因产品的短期表达成为可能。在最近的研究中，NILV被证明能在体内有效地诱导抗原特异性免疫反应，目前正被用作传递锌指核酸酶的平台，以介导定点基因编辑。然而，需要对NILV系统进行进一步的改进，以使这种有希望的基因输送系统适合于人类临床试验。这些改进包括：a)增加外体基因的表达，b)减少非法载体整合，c)开发有效的非免疫原性基因调控系统，d)建立高效的载体生产系统。为了实现这些改进，我们在这里概述了一个由四个目标组成的研究方案。在目标1中，我们建议开发一种新的NILV，该NILV删除了宿主LSF-YY1和AP-4转录抑制物识别的顺式抑制元件。这一拟议的修饰将缓解NILV典型的转录沉默，并将提高它们的效力。目标2将专注于开发一种以选择性剪接为前提的新型可诱导NILV系统。与目前使用的诱导系统不同，新型剪接调控的NILV不包含合成的反式激活剂，这可能会诱导细胞介导的免疫反应。在目标3中，我们将集中于一种新型的多尿路(PPT)缺失的NILV的特征和体内测试，该NILV表现出减少的非法整合。在最后一个目标中，我们将建立第一个整合酶缺乏的稳定包装细胞系，它将产生具有目标1-3中所述改进的高滴度近ILV。所有包含不同修饰的新载体都将携带改进的人凝血因子IX(HFIX)cDNA.这些载体将通过瞬时转基因或由新的稳定包装细胞系产生，它们治愈Fix缺陷的能力将在血友病B小鼠模型中进行测试。

DESCRIPTION (provided by applicant): Nonintegrating lentiviral viral vectors (NILVs) present a means of reducing the risk of insertional mutagenesis in nondividing cells and enabling the short-term expression of potentially hazardous gene products. In recent studies, NILVs proved efficient at inducing an antigen specific immune response in vivo and are currently being used as a platform to deliver zinc finger nucleases to mediate site-specific gene editing. However, additional improvements in the NILV system are required to render this promising gene delivery system suitable for human clinical trials. These improvements include: a) increasing episomal gene expression, b) reducing illegitimate vector integration, c) developing an efficient and nonimmunogenic gene regulation system, and d) establishing an efficient vector production system. To achieve these improvements, we outline here a research proposal consisting of four aims. In aim 1 we propose to develop a novel NILV deleted of the cis inhibitory elements recognized by the host LSF-YY1 and AP-4 transcriptional repressor complexes. This proposed modification will alleviate the transcriptional silencing typical of NILVs and will improve their efficacy. Aim 2 will focus on the development of a novel inducible NILV system premised on alternative splicing. In contrast to currently used inducible systems, the novel splicing-regulated NILVs do not contain a synthetic transactivator, which could potentially induce a cell-mediated immune response. In aim 3, we will focus on characterization and in vivo testing of a novel polypurine tract (PPT) deleted NILV, which exhibits reduced illegitimate integration. In the last aim, we will establish the first integrase-deficient stable packaging cell line, which will produce high-titer NILVs bearing the improvements described in aims 1-3. All new vectors containing the different modifications will carry an improved human factor IX (hFIX) cDNA. The vectors will be produced either by transient transfection or by the novel stable packaging cell line, and their ability to cure FIX deficiency will be tested in a hemophilia B mouse model.